Article

Glycoconjugate Journal

, Volume 25, Issue 3, pp 245-257

Open Access This content is freely available online to anyone, anywhere at any time.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

  • Véronique BlanchardAffiliated withBijvoet Center, Department of Bio-Organic Chemistry, Utrecht UniversityInstitute of Clinical Chemistry and Pathobiochemistry, Charité University
  • , Rupali A. GadkariAffiliated withDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science
  • , Albert V. E. GeorgeAffiliated withBijvoet Center, Department of NMR Spectroscopy, Utrecht University
  • , Satarupa RoyAffiliated withDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science
  • , Gerrit J. GerwigAffiliated withBijvoet Center, Department of Bio-Organic Chemistry, Utrecht University
  • , Bas R. LeeflangAffiliated withBijvoet Center, Department of Bio-Organic Chemistry, Utrecht University
  • , Rajan R. DigheAffiliated withDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science
  • , Rolf BoelensAffiliated withBijvoet Center, Department of NMR Spectroscopy, Utrecht University
  • , Johannis P. KamerlingAffiliated withBijvoet Center, Department of Bio-Organic Chemistry, Utrecht University Email author 

Abstract

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Keywords

Pichia pastoris GS115 Pichia pastoris X-33 Human chorionic gonadotropin Human follicle stimulating hormone Glycosylation Hypermannosylation 15N-Labeling